Nanopore forming method and uses thereof

ABSTRACT

The invention relates to a method for making nanopores in thin layers or monolayers of transition metal dichalcogenides that enables accurate and controllable formation of pore within those thin layer(s) with sub-nanometer precision.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/432,163 filed Jun. 5, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/688,264 filed Aug. 28, 2017, now U.S. Pat,. No.10,364,507, which is a continuation of PCT Application No.PCT/M2016/051425 filed Mar. 12, 2016, which claims the benefit ofpriority to European Patent Application No. 15158894.4, entitled“NANOPORE FORMING METHOD AND USES THEREOF,” filed Mar. 12, 2015;European Patent Application No. 15171077.9, entitled “NANOPORE FORMINGMETHOD AND USES THEREOF,” filed Jun. 8, 2015; and U.S. ProvisionalPatent Application No. 62/286,235, entitled “NOVEL METHOD FOR NANOPOREFORMATION IN ULTRATHIN MEMBRANES SUITABLE FOR MANUFACTURE,” filed Jan.22, 2016, the contents of all of which are incorporated herein byreference for all purposes.

FIELD OF THE INVENTION

The present invention pertains generally to the fields of nanoporeforming in ultrathin membranes based on two-dimensional materials, inparticular for use in molecular sensing devices, more particularlysolid-state sensing of biomolecules such as DNA, RNA and proteins.

BACKGROUND OF THE INVENTION

Solid state nanopore bio-sensing is emerging as a rapid single moleculesensing technique (Branton et al., 2008, Nature Biotechnology, 26, 1146;Dekker, 2007, Nature Nanotechnology 2, 209). Conceptually, a singlenanometer size aperture located on a membrane can detectelectrophoretically driven biomolecules translocation in a highthroughput manner, revealing localized information of the analyte.However, the formation of single nanopores relies heavily on expensiveinstrumentation, i.e., Transmission Electron Microscope (TEM) and welltrained TEM user, which renders it still confined to laboratory usesince this nanopore fabrication process is time-consuming, expensive,not scalable and hard to control at the nm scale.

Further, high costs of the TEM use, coupled with its high initialinvestment and the time consuming pore drilling process (1 hour machineand operator time per device) limit the more extensive application ofsolid state nanopores in the bio-sensing field. In addition, not all

TEM drilled nanopores are hydrophilic and functional for the sensing ofbiomolecules. In addition, interaction with the high energy electronbeam can cause damage especially when dealing with membranes in 2 Dmaterials.

Many efforts, such as chemical wet-etching of silicon (Park et al.,2007, Small 3, 116-119) or polyethylene terephthalate film (Siwy et al.,2002, Physical review letters 89, 198103) have been carried out towardsmass production of nanopores. Recently, a facile method has beenreported using dielectric breakdown to make individual nanopores (3-30nm diameter) on insulating silicon nitride membranes (5-30 nm thick)without the need of TEM_ENREF_8 (Kwok et al., 2014, Plos One 9,doi:10.1371/journal.pone.0092880, WO 2013/167952) for the in-situforming of nanopores. However, those techniques based on dielectricbreakdown need to apply high voltages pulses to the membranes whichshould be as short as possible for trying to monitor the nanoporediameter during its formation (WO 2014/144818). When reaching dielectricbreakdown, the process of pore forming becomes rather uncontrollablewhich is problematic for reproducibility and quality control of thenanopore size, especially when formed in-situ in a nanopore bio-sensingdevice, thereby leading to important production waste if the quality ofthe pore does not correspond in fine to the prescribed parameters.

Atomically thin nanopore membranes, graphene (Garaj et al., 2010,Nature, 467, 190) and molybdenum disulphide (MoS₂) (Feng et al., 2015,Nature Nanotechnology, 10, 1070) have drawn much attention recently dueto their unprecedented single nucleotide resolution and holds promise asa candidate for so called 3^(rd) generation DNA sequencers. Therefore,if fabrication of nanostructures with sub-nanometer, or even single-atomprecision has been a long-term goal for nanotechnology in general, thereis now a raise of interest for those thin nanopore membranes and anincreased need for cost-effective and reliable techniques for nanoporeformation in those membranes.

In particular, since the differentiation of biomolecules relies stronglyon the pore diameter, there is a high need for developing methodsallowing controllable nanopore fabrication, which would enable massproduction nanopore in 2D membranes such as MoS₂ even below 4 nm withatomic precision.

BRIEF SUMMARY OF THE INVENTION

An object of this invention is to provide a method for making nanoporesin thin nanopore membranes of transition metal dichalcogenide thatenables accurate and controllable formation of pore with sub-nanometerprecision.

It is advantageous to provide a method of nanopore formation where thepore formation can be carried out in situ, notably in a nanoporebio-sensing device.

It is advantageous to provide a method of nanopore formation where thesize of the pore is monitored during pore formation and adapted ondemand, depending on the sizing need for the different applications suchas the type of biomolecules to be sensed (e.g. proteins, or DNA-proteincomplexes, ss-DNA, dsDNA, RNA polymers, nucleotides, Nucleic Acidsurrogates, or any non-natural polymers DNA tags).

It is advantageous to provide a system for nanopore formation that iseconomical to implement for mass production and easy to use.

It is advantageous to provide a system for nanopore formation thatallows the formation of a broad series of nanopore in parallel ondifferent membranes.

Another object of this invention is to provide method for forming ananopore in an electrochemically etchable 2D material.

Disclosed herein, according to a first aspect of the invention, is amethod for forming a nanopore in a membrane of transition metaldichalcogenide (TMDC) crystals comprising the steps of:

-   -   providing a TMDC thin layer having from about 0.3 nm to 2 nm        thickness (Hm) immersed in an electrically conducting liquid ;    -   applying a transmembrane voltage (V) at a value higher than the        oxidation potential of the transition metal of the said TMDC to        the said TMDC thin layer;    -   measuring the ionic current (I_(i)) in the said electrically        conducting liquid;    -   turning off the transmembrane voltage once the measured ionic        current (I_(i)) has reached a value (I_(p)) corresponding to the        electrical conductance of a pore in the TMDC thin layer having a        prescribed diameter (d_(p)).

The applied transmembrane voltage is preferably an essentially constantDC voltage applied between electrodes located on opposing sides of theTMDC thin layer.

In an embodiment, the transition metal dichalcogenide (TMDC) is ofchemical formula is MX₂, where M is a transition metal atom and X is achalcogen (S, Se, or Te).

In a particular embodiment, M is a transition metal atom selected fromTa, Nb, Mo, W, Ti and Re.

According to a particular embodiment, the TMDC is selected from MoS₂,SnSe₂, WS₂, TaS₂, MoSe₂, WSe₂, and TaSe₂.

According to another particular embodiment, the TMDC is selected fromNbS₂, NbSe₂, TiS₂, TiSe₂, ReS₂ and ReSe₂

According to a particular embodiment, the TMDC is MoS₂.

According to another particular embodiment, the TMDC is WSe₂.

According to a particular aspect, the TMDC thin layer can be employed insingle, double or multilayer form.

According to a further particular aspect, the TMDC thin layer may be amonolayer.

According to a particular embodiment, the TMDC thin layer is suspendedin an electrically conducting liquid in such a way that a portion of thethin layer extends over a support layer and the other portion of thethin layer is in contact of a support layer.

In an embodiment of the invention, the material of the support layer maycomprise SiN_(x), glass, Al₂O₃ or HfO₂.

In another embodiment of the invention, the material of the supportlayer may comprise quartz, or TiO₂.

According to another further particular embodiment, the membrane oftransition metal dichalcogenide (TMDC) crystals is a single-layer.

According to an advantageous embodiment, the TMDC layer comprises MoS₂thin layers or is a MoS₂ monolayer.

According to an advantageous embodiment, the TMDC layer comprises CVDgrown thin layers or is a CVD grown monolayer.

According to another further embodiment, the TMDC layer comprises MoCVDgrown thin layers or a MoCVD grown monolayer.

In an embodiment of the invention, the electrically conducting liquidmay advantageously comprise or consist in an aqueous liquid comprisingan electrolyte.

In an embodiment of the invention, the electrically conducting liquidmay be different from each side of the transition metal dichalcogenidemembrane, in particular when the nanopore formation process is to beconducted in situ in a nanopore sensing device.

In an embodiment of the invention, the electrolyte may be potassiumchloride (KCl).

In an embodiment of the invention, the electrically conducting liquid isan aqueous ionic solution (e.g. water and KCl or any inorganic saltssuch as LiCl, NaCl, MgCl₂, CaCl₂ etc).

In another embodiment, the transmembrane voltage is applied in acontinuous manner.

In another embodiment, the transmembrane voltage is applied for a periodof time necessary to reach an ionic current value which corresponds tothe electrical conductance of a pore having a prescribed diameter(prescribed current/conductance).

In an embodiment of the invention, the ionic current can be measured inan ionic current circuit comprising a pair of electrodes (e.g. Ag/AgClor Pt) coupled to the conducting liquid on opposite sides of themembrane.

In an embodiment, the ionic current circuit comprises means to measurethe ionic current configured to provide a signal used in the measurementof the conductance through the nanopore.

According to a particular embodiment, the transmembrane voltage isswitched off once the measured ionic current (I_(i)) has reached a value(I_(p)) corresponding to the electrical conductance of a pore in theTMDC thin layer having a prescribed diameter (d_(p)).

According to another embodiment, the turning off of the transmembranevoltage is achieved by an automatic switch which is activated through afeed-back control circuit when a prescribed current/conductance isreached.

According to a particular embodiment, the turning off of thetransmembrane voltage is achieved by a decrease of the transmembranevoltage to a value (Vd), wherein the value (Vd) is 50% of the voltage atwhich electrochemical reaction occurs for a given pore and experimentalcondition or lower than 50%, wherein the decrease is initiated once themeasured ionic current (I_(i)) has reached a value (I_(p)) correspondingto the electrical conductance of a pore in the TMDC thin layer having aprescribed diameter (d_(p)). Typically, the said transmembrane voltageis decreased to a value (V_(d)) from about 800 mV-2V to about 50%-90%lower than said oxidation potential.

According to another embodiment, the decrease of the transmembranevoltage is achieved by an automatic variator which is activated througha feed-back control circuit when a prescribed current/conductance isreached.

According to another embodiment, the oxidation potential of a transitionmetal dichalcogenide can be determined by cyclic voltametry. Forexample, a voltage of 800 mV is higher (typically between 800 mV and1,000 mV such as about 800 mV and 900 mV) than the oxidation potentialof MoS₂ being oxidized to Mo^((VI)) and allows starting the ECR process.

For example, in the case of the transition metal dichalcogenide beingWSe₂, a voltage of 1V is higher (typically between 1,000 mV and 1,200 mVthan oxidation potential of WSe₂ being oxidized to Wm and allowsstarting the ECR process.

For another example, in the case of the transition metal dichalcogenidebeing WSe₂, a voltage of 1V is higher (typically between 1000 mV and2000 mV than oxidation potential of WSe₂ being oxidized to Wm and allowsstarting the ECR process.

According to a particular embodiment, once the ECR process starts, thetransmembrane voltage is applied at a potential slightly lower thanoxidation potential for the bulk material, for example about 5% lowerthe oxidation potential (e.g. about 5% (±1 or 2%) lower). This allowsthe manufacture of a single pore at a vacancy instead of making severalpores outside such vacancy.

According to a particular embodiment, the transmembrane voltage isapplied at a potential slightly higher than oxidation potential (e.g.about 5 to 10% higher) and once the ECR process starts, thetransmembrane voltage is applied at a potential slightly lower thanoxidation potential for the bulk material, for example about 5±1 or 2%lower the oxidation potential.

According to another embodiment, is provided a method of manufacturing ananopore sensing device comprising a step of forming a nanoporeaccording to the invention.

According to a further embodiment, is provided a method of manufacturinga nanopore sensing device according to the invention further comprisinga step, once the nanopore is formed, of exchanging the electricallyconducting liquid in the cis side of the transition metal dichalcogenidemembrane by an electrically conducting liquid. According to a particularaspect, this electrically conducting liquid comprises a room temperatureionic liquid (RTIL) or an aqueous ionic solution such as water and KClor any inorganic salts such as LiCl, NaCl, MgCl₂ or CaCl₂.

In a further embodiment of the invention, the room temperature ionicliquid (RTIL) is selected from an essentially pure RTIL, optionallymixed with an organic solvent, or a mixture of a water-miscible RTIL inwater with a water content from about 5 to about 50 wt %.

According to an embodiment, is provided a method according to theinvention, wherein more than one TMDC thin layers are provided and atransmembrane voltage is applied to each of the TMDC thin layers inparallel.

Disclosed herein, according to a first aspect of the invention, is amethod for forming a nanopore in an electrochemically etchable 2Dmaterial comprising the steps of:

-   -   providing at least one thin layer of said electrochemically        etchable 2D material having from about 0.3 nm to 5 nm thickness        (Hm) suspended in an electrically conducting liquid;    -   applying a transmembrane voltage (V) at a value slightly higher        than the oxidation potential of a transition metal of said at        least one electrochemically etchable thin layer configured for        an electrochemical atomic etching of said thin layer;    -   measuring the ionic current (I_(i)) in the said electrically        conducting liquid;    -   turning off the transmembrane voltage once the ionic current        (I_(i)) has reached a value (I_(p)) corresponding to the        electrical conductance of a pore within the said TMDC thin layer        having a prescribed diameter (d_(p)).

In a further embodiment of the invention, the electrochemically etchable2D material is a membrane of transition metal dichalcogenide (TMDC)crystals or any electrochemically etchable 2D material such as hBnsilicene.

In another further embodiment of the invention, the electrochemicallyetchable 2D material has a thickness of about 0.3-1.5 nm thickness (Hm).

In another further embodiment of the invention, the electrochemicallyetchable 2D material is a material as described in Nicolosi et al.,2013, Science, 340 (6139), DOI: 10.1126.

In a further embodiment of the invention, the electrochemically etchable2D material is a membrane of transition metal dichalcogenide (TMDC)crystals or any electrochemically etchable 2D material selected from agroup comprising hBn silicene, transition metal trichalcogenides, metalhalides, transition metal oxides and the like.

The above mentioned features may be combined in any appropriate manner.

An advantageous characteristic of the invention is to provide a methodwhere the pore characteristics such as size, shape, and edge propertiesare fully controllable and reproducible.

An advantageous characteristic of the invention is to provide a methodwhere the pore formation process lasts for few minutes of less.

An advantageous characteristic of the invention is to provide a methodwhere the formation of the nanopore(s) in the transition metaldichalcogenide membrane is achieved by atomically controlledelectrochemical etching of transition metal dichalcogenide thin layer,including monolayers with a sub-nanometer precision

According to one aspect, the method of the invention allows in-situpreparation of nanopores in membranes of a transition metaldichalcogenide by electrochemical reaction (ECR), in particular in ananopore sensing device.

An advantageous characteristic of the invention is to provide a methodwhere pore dimensions may be adjusted to the needs, in particular to thebiomolecule to sense in a nanopore sensing device.

A noticeable advantage for a nanopore fabrication method of theinvention is that biomolecule translocations can be performed in situdirectly after ECR and size-control allows on-demand adaptation of thepore size, allowing sizing for the different types of biomolecules, e.g.proteins or DNA-protein complexes etc.

According to a further aspect, the method can be applied to a pluralityof transition metal dichalcogenide membranes using a multiple channelsfeedback control unit for monitoring the ionic current through aplurality of transition metal dichalcogenide membranes.

Referring to FIG. 8, is illustrated a method for forming a nanopore in atransition metal dichalcogenide membrane according to the inventionwherein the transition metal dichalcogenide membrane is in a form of athin layer (or a monolayer) 1 having from about 0.3 nm to 4 nm thicknessas depicted in FIG. 1A wherein more than one TMDC thin layers (1,1′ and1″) are provided and a transmembrane voltage is applied to each of theTMDC thin layers in parallel (V, V′ and V″). Each TMDC thin layer issuspended in an electrically conducting liquid (2, 2′, 2″) and eachtransmembrane voltage is applied at a value higher than the oxidationpotential of the transition metal of the corresponding TMDC, measuringthe ionic current (I_(i)) in the said electrically conducting liquiduntil the ionic current (I_(i)) has reached a value (I_(p))corresponding to the electrical conductance of a pore within the metaldichalcogenide membrane having a prescribed diameter (D_(p)).

The TMDC thin layer 1 is configured such that the portion of the TMDCthin layer where the formation of a nanopore will be carried out issuspended in an electrically conducting liquid and the other portion ofthe TMDC thin layer is supported by a support layer (3, 3′ and 3″).

A multiple channels feedback control unit 7 (e.g. N channel FGPA card)is used for achieving in parallel (a) monitoring transmembrane voltagethrough each of the TMDC thin layers, (b) monitoring the recording ofthe measurement of the ionic current in an electrical circuit (5, 5′,5″) formed between a voltage source V (V′, V″) and electrodes 6 a and 6b (6 a″, 6 b″; 6 a″, 6 b″) immerged into the electrically conductingliquid on both sides of the corresponding TMDC thin layer 1, 1′, 1″ and(c) the turning off of the transmembrane voltage when a prescribedcurrent/conductance is reached for the corresponding TMDC thin layer.The different thin layers (1, 1′ and 1″) can be provided in the form ofthin membrane devices M1 to Mn mounted in parallel. A preamplifying unitfor each of electrical circuit (6, 6′, 6″) can be connected to themultiple channels feedback control unit 7 and the voltage sources.

According to an embodiment, by for instance using a field programmablegate array (FPGA) board (NI PXI-7851R), the manufacture of a pluralityof nanopores, for instance eight, can be monitored in parallel.

Moreover, a method according to the invention could also pave the way tocheaper sensing devices by taking advantage of wasteless in situnanopore forming adapted for scale up production of 2D nanopores andshrink the costs for sensing devices based on such nanopore membranes.

Embodiments described herein may provide for improved methods andsystems of forming nanopores and for detecting biological molecules inthe nanopores. Embodiments include nanopores in solid state materials,which may allow for more consistent and easily fabricated structures.The solid state materials may allow for sub-nanometer or atomic levelcontrol of a nanopore formed within the materials. These nanopores maybe fabricated in thin film materials, which when used to analyze anucleic acid molecule, may allow only 1 to 3 bases in the nanopore at atime. This small number of bases may then increase the precision ofdetecting or analyzing biological molecules. Analyzing biologicalmolecules may include single molecule nucleic acid sequencing.Furthermore, a thinner height of the material in which the nanopore isformed may facilitate wetting of the pores.

Embodiments may include a method of forming a nanopore having a desireddiameter. The method may include applying a current having a constantamplitude and a constant frequency to a first liquid. The first liquidmay be on one side of a layer of transition metal dichalcogenidecrystals. The method may also include applying the current to a secondliquid. The second electrically conductive liquid may be on another sideof the layer of transition metal dichalcogenide crystals. Additionally,the method may include widening an aperture in the layer of transitionmetal dichalcogenide crystals until the nanopore having the desireddiameter is formed.

In some embodiments, methods may include a method of forming a nanoporehaving a desired diameter. The method may include applying a voltageacross a layer of transition metal dichalcogenide crystals. The layer oftransition metal dichalcogenide crystals may have a first liquid on oneside and a second liquid on another side. The method may also includewidening an aperture in the layer of transition metal dichalcogenidecrystals. The method may further include reducing the voltage to stopwidening the aperture to form the nanopore having the desired diameter.

Embodiments may include a nanopore device for analyzing biologicalmolecules. The nanopore device may include a layer of transition metaldichalcogenide crystals. The layer of transition metal dichalcogenidecrystals may define an aperture. The nanopore device may also include aninsulating material contacting the layer of transition metaldichalcogenide crystals. The device may further include a liquid on bothsides of the layer of transition metal dichalcogenide crystals. Inaddition, the nanopore device may include two electrodes configured toapply a current to the liquid or a voltage across the electrodes. Bothelectrodes may be in the liquid. The components of the nanopore devicemay allow the nanopore device to detect and/or analyze biologicalmolecules that enter the nanopore.

These and other embodiments may also include a device for analyzingbiological molecules. The device may be created by any of the methodsdescribed herein.

Some embodiments may include a method of analyzing biological molecules.The method may include using any device described herein. The method mayinclude translocation of a biological molecule through the nanopore. Thebiological molecule may be a biological polymer or a monomer derivedtherefrom.

Other features and advantages of the invention will be apparent from theclaims, detailed description, and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are illustrations of exemplary settings for carrying outthe process of the invention. FIG. 1A shows disposition of the 2Dtransition metal dichalcogenide for the in situ formation of a nanopore.FIG. 1B is a flowchart illustration of the steps of a process of theinvention as implemented according to an exemplary embodiment of Example1.

FIGS. 2A-E are illustrations of the use of a method of the invention forin situ fabricating nanopores in a transition metal dichalcogenidemembrane in a molecular sensing system as described in Example 1. FIG.2A is a schematic illustration of preparation of a freestanding MoS₂membrane ready for electrochemical formation of a nanopore. In thecenter of the supporting 20 nm thick SiN_(x) membrane a single focusedion beam, focused ion beam (FIB) hole is drilled to suspend a smallportion of an intact monolayer MoS₂ flake. A single chip is mounted inthe flow-cell for typical translocation experiments. A pair ofelectrodes (e.g. Ag/AgCl) is connected to a preamplifier is used toapply transmembrane voltage. FIG. 2B shows an optical image of theSiN_(x) membrane with a FIB drilled hole in the center. FIG. 2C shows anoptical image of the SiN_(x) membrane with transferred triangularCVD-grown MoS₂ monolayer. FIG. 2D shows a low magnification TEM image oftransferred CVD-grown MoS₂ monolayer covering the FIB hole. The FIB holeis indicated by the black arrow. FIG. 2E shows a high resolution TEM ofthe lattice of MoS₂ suspended over the FIB hole. The correspondingdiffraction diagram is shown in the inset.

FIGS. 3A-E show the leakage current characteristics of an intactmembrane and during pore formation as described in Example 1 togetherwith the visualization and modelization of the pore forming process.FIG. 3A shows a leakage current-voltage (I-V) characteristic of anintact MoS₂ membrane, for voltages below the critical voltage of 800 mVrequired for ECR which depends on the number of the membrane defects(more defects leads to higher current). FIG. 3B shows a representativeionic current trace measured for a MoS₂ membrane. Voltage is stepped by100 mV with a 50 s holding, and the leakage current increases inaccordance, being steady for a constant voltage. Sharp peaks at eachvoltage step originate from the capacitance charging. After a criticalvoltage, 800 mV is applied, the electrochemical reaction (ECR) starts(arrow), the current keeps increasing which triggers the feedbackcontrol to switch off voltage bias in order to halt the pore growth.FIG. 3C shows a mechanism of ECR based MoS₂ nanopore fabrication. A sideview of the monolayer MoS₂ lattice, emphasizing the lattice havingsingle atom (S) vacancy or defect before ECR V<Vcritical (left), MoS₂lattice at V=Vcritical (nanopore starts to form, middle) and MoS₂lattice when nanopore (diameter d) is formed (right). FIG. 3D shows acurrent-voltage (IV) characteristic of nanopores ranging in diameterfrom 1 to 20 nm—all nanopores are created via electrochemical reaction.Inset shows I-V characteristics for the system below and at the criticalvoltage. FIG. 3E shows TEM images verifying the nanopore formation andsize (top being a zoom-in image of the bottom image).

FIGS. 4A-D show simulations of the electric potential distribution forthe nanopore in two dimensions for a freshly formed pore having adiameter of 0.3 nm and comparison with a typical current trace ofnanopore formation on graphene membrane. FIG. 4A shows electricpotential distribution in the trans chamber in the immediate vicinity ofthe membrane surface and (FIG. 4B) in the cis chamber. FIG. 4C showselectric potential distribution as a function of the distance from thepore. The applied potential was set to 800 mV and salt concentration was1 M KCl. FIG. 4D shows typical current trace of nanopore formation ongraphene membrane using ECR.A much higher transmembrane voltage, 2.8 Vhas to be applied to graphene to create a nanopore in graphene.

FIGS. 5A-E are schematization of nanopore forming in monolayer MoS₂starting from a monolayer MoS₂ lattice. FIG. 5A is a top view of themonolayer MoS₂ lattice, the unit cell, u (parameter a=3.12 Å) is shownin grey (Hulliger, & Lévy. Structural Chemistry of Layer-Type Phases,Springer, 1976, p. 236). FIG. 5B shows ionic current-steplike featuresduring the nanopore formation in FIG. 2A. FIG. 5C shows custom Matlabcode is used to detect steps in the raw ionic current trace (Raillon etal., 2012, Nanoscale, 4, 4916-4924 and histogram of the trace shown inb) with corresponding color coded atom groups cleaved in each stepduring the pore formation from 1 to 21. FIG. 5D is an illustrativeschematic of polygon removal corresponding to the histogram trace.Aberration corrected TEM micrograph of suspended single layer MoS₂superimposed are the polygons which correspond to atom groups cleaved inthe steps 1, 7, 14 and 21 during the pore formation. The coloring ofatom groups cleaved in each step (FIG. 5C) and corresponding areapolygons shown in the FIG. 5D starts from step 1 and goes up to step 21.In FIG. 5E, the table represents the sequence of cleaving MoS₂ unitcells and Mo and S atoms in 21 steps to form the pore. I_(step) is thedistance between two adjacent peaks in the current histogram; D and Aare effective pore diameter and pore area, respectively; N is the numberof unit cells equivalent to the effective area; ΔA is the increments ofA; ΔN is the increments of N; ΔX is the nearest integer or integer +⅓ or+⅔ of ΔN; the last column on the right stands for the number of unitcells.

FIGS. 6A-B show DNA translocation through a nanopore fabricated asdescribed in Example 1. FIG. 6A is a typical trace of pNEB plasmid DNAtranslocation through an electrochemically etchhed nanopore recorded at450 mV. The trace is downsampled to 10 kHz for display. FIG. 6B is ascatter plot of events collected at 300 mV and 450 mV bias. Eventdetection is performed using OpenNanopore37 Matlab code. Expectedly, theincrease in the bias voltage from 300 to 450 mV shortens thetranslocation time and enhances the current drop.

FIG. 7 shows selected events of λ-DNA translocation through a 4 nm ECRfabricated MoS₂ nanopore by a method according to the invention asdescribed in Example 1 and recorded in-situ right after pore formationat 200 mV as described in Example 2. Quantized levels in the ioniccurrent represent the flexibility of λ-DNA when translocating throughnanopore.

FIG. 8 shows and example of arrangement of setting for carrying out theprocess of the invention for the in situ formation of a nanopore onseveral devices comprising a 2D transition metal dichalcogenide membranein parallel through the control of an integrated circuit configured forthe monitoring of the application and cut-off (e.g. N-channel FPGA card)transmembrane voltages in parallel to each of the 2D transition metaldichalcogenide membranes.

FIGS. 9A-C illustrate the process of the invention carried out on anexfoliated single layer of WSe₂. FIG. 9A shows an exfoliated singlelayer of WSe₂transferred to silicon nitride membrane and positioned overthe small opening on the same device layout as MoS₂ as described inExample 1. FIG. 9B shows current voltage characteristics of nanoporeformed with ECR in WSe₂ when critical voltage of 1V reached. FIG. 9C isa representative ionic current trace measured for WSe₂ membrane for avoltage set to the critical voltage of 1V where the current keepsincreasing until desired pore size is reached.

FIGS. 10A-D show an apparatus for forming a nanopore before and after anaperture is opened according to embodiments of the present invention.

FIG. 11 shows a block flow diagram of a method of forming a nanoporeusing an applied variable voltage with constant current according toembodiments of the present invention.

FIG. 12 shows a block flow diagram of a method of forming a nanoporeusing an applied voltage with variable current according to embodimentsof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Certain conventional methods of forming nanopores for detection oranalysis of biological molecules may have certain disadvantages.Nanopores may be formed in protein layers, but protein layers may needto be inserted in a lipid bilayer. Lipid bilayers are often fragile, andmethods of making nanopores in protein layers in lipid bilayers may havelow yields. Additionally, the atomic and molecular moieties at thesurface of a protein nanopore may not be known.

Some conventional solid state nanopores may present other problems.Extremely thin layers of materials such as silicon oxides and siliconnitrides may be difficult to deposit. These layers may be 5 to 10 nm inthickness, resulting in space for 50 to 100 bases in a single nanoporeat the same time. Materials, such as silicon dioxide, may be prone tocontamination from carbon and other compounds. Additionally, creating ananopore with a target diameter may be difficult in these materials.

A material such as graphene may also not be ideal for forming nanopores.Graphene sheets are typically 1 Å thick, which may be too thin fordetection or analysis of biological molecules. A biological moleculeinside a graphene nanopore may not generate enough of a change in ioniccurrent for accurate and precise detection or analysis. Graphene mayalso be conductive, which may generate interference when measuringcurrent through the graphene.

Nanopores in a layer of transition metal dichalcogenide crystals mayallow for improved fabrication and detection/analysis. Transition metaldichalcogenide crystals may not corrode easily. Additionally, a thinlayer of transition metal dichalcogenide crystals may be formed,allowing for 1 to 3 bases to be in a nanopore at the same time.Furthermore, because the layer is crystalline, the atoms and chemistryat the surface of the nanopore and the rest of the layer is known.Conventional methods of forming nanopores often use a beam of ions orelectrons, while methods described herein may not use such beams andinstead use a pair of electrodes. Methods for forming nanopores andnanopore devices for detection or analyzing molecules are describedherein.

Referring to the figures, in particular first to FIGS. 1A and 2A-E, amethod for forming a nanopore in a transition metal dichalcogenidemembrane, in a form of a thin layer (or a monolayer) 1 having from about0.3 nm to 4 nm thickness (Hm) suspended in an electrically conductingliquid 2 comprises applying a transmembrane voltage (V) at a valuehigher than the oxidation potential of the transition metal of the saidTMDC, measuring the ionic current (I_(i)) in the said electricallyconducting liquid until the ionic current (I_(i)) has reached a value(I_(p)) corresponding to the electrical conductance of a pore within themetal dichalcogenide membrane having a prescribed diameter (D_(p)).

The TMDC thin layer 1 is configured such that the portion of the TMDCthin layer where the formation of a nanopore will be carried out issuspended in an electrically conducting liquid and the other portion ofthe TMDC thin layer is supported by a support layer 3.

The support layer can be of SiN_(x), glass, quartz, Al₂O₃, or HfO₂ (orany other material that provides low capacitance membrane) with asupport orifice of a diameter Ds that allows the portion of the TDMCmembrane where the pore formation should be conducted being suspended inthe electrically conducting liquid.

When the transmembrane voltage V is applied to the TMDC thin layer at avalue higher than the oxidation potential of the transition metal of thesaid TMDC, TMDC start being oxidized through an ECR reaction and theformation of a nanopore 4 is initiated.

The formation for a nanopore 4 could be monitored through themeasurement of an increase in the ionic current (I_(i)) in an electricalcircuit 5 formed between a voltage source V and electrodes 6 a and 6 bimmerged into the electrically conducting liquid on both sides of theTMDC thin layer 1, and configured to measure the ionic current L by acurrent measurement circuit portion A. The current measurement circuitportion A measures the ionic current value which is representative ofconductance through the pore in the TMDC thin layer 1 and therefore ofthe diameter D_(p) of the pore 4 formed in the TMDC thin layer 1.

Thin layers of MoS₂ with good quality suitable for use in a deviceaccording to the invention can be prepared by both exfoliation andchemical vapor deposition (CVD) (Novoselov et al., PNAS, 2005, 102,10541-1053; Liu et al. 2012, Nano Lett., 12, 1538-1544).

Typically, the thickness of TDMC membranes according to the inventioncan be assessed by Raman/optical electron microscopy, photo-luminescence(PL) measurements and Atomic Force Microscopy (AFM).

According to another particular aspect, the thickness of a TDMC membraneaccording to the invention may be less than 2 nm, typically from about0.7 nm to less than 2 nm. In particular, the TDMC membrane is from about0.7 nm (e.g. one layer) to about 1.4 nm thick (e.g. two layers).

According to another aspect, pores in the TDMC membrane formed by aprocess of the invention are nanometer sized, typically from about 1 nmto 20 nm diameter (for example typically from about 1 nm to about 5 nm,for example less than 4 nm or less such as about 3 nm) and from about0.3 nm to 1 nm thickness (for example about 0.7 nm). Typically, the sizeof the pores can be measured by Transmission electron microscopy (TEM)and calculated from the current-voltage characteristics.

According to another aspect, the support layer can be a SiN_(x), glass,or quartz (or any other material that provides low capacitance membrane)with a support orifice that allows the portion of the TDMC membranewhere the pore formation should be conducted being suspended in theelectrically conducting liquid. According to a further aspect, thesupport orifice has typically a diameter of from about 20 nm to about500 nm (e.g. 50 nm), like for example from about 200 nm to about 500 nmand from about 20 nm to 50 nm thick.

According to a further aspect, the support layer can be coated with somecuring layer such as polydimethylsiloxane (PDMS), while leaving the MoS₂nanopore exposed in order to reduce the dielectric noise. Alternatively,support layer can be a quartz, glass, or any other material thatprovides low capacitance membrane based support.

According to a particular embodiment, the TDMC thin layer of theinvention can be an active layer as described in WO 2012/093360.

According to a particular embodiment, the method of the invention can becarried out in a nanopore sensing device as generally described inPCT/EP2015/053042.

Referring to the method according to the invention, it has to beunderstood that the expression “turning off the transmembrane voltage”should be understood as encompassing the complete switching off thetransmembrane voltage or alternatively the decrease of the transmembranevoltage to a value V_(d) which is 50% of the voltage at whichelectrochemical reaction occurs for a given pore and experimentalcondition (pH, buffer composition, etc.) or lower than 50% of saidvoltage in order to stop the pore formation.

Referring to FIG. 1B, provided is an illustration of a specificembodiment regarding steps of a method for forming a nanopore in atransition metal dichalcogenide membrane according to the invention:

-   -   (a) providing a TMDC thin layer mounted in a housing (e.g.        polymethylmethacrylate (PMMA)) flow cell chamber on a support        layer (e.g. SiN_(x)) with a support orifice that allows the        portion of the TDMC membrane where the pore formation will be        conducted being suspended in the electrically conducting liquid,        wherein the housing comprises a cis and a trans chamber and the        TMDC thin layer is located at the interface of those two        chambers;    -   (b) filling cis and trans chambers with an aqueous solution        (e.g. a mixture of ethanol/deionized water v/v, 1:1) for        increasing its hydrophilicity (e.g. for about 15-35 min);    -   (c) flushing the cis and trans chambers with an electrically        conducting liquid (e.g. 1M KCl) such as the TMDC thin layer is        immersed in said electrically conducting liquid;    -   (d) immersing a pair of electrodes adapted to the electrically        conducting liquid (e.g. freshly made Ag/AgCl electrodes) in the        corresponding chambers and connected to a voltage source, for        example through a programmed amplifier;    -   (e) applying a transmembrane voltage by step-wise increments        (e.g. 20-100 mV increments) and holding each step for about        25-50 s;    -   (f) measuring the ionic current (I_(i)) in the said electrically        conducting liquid;    -   (g) turning off the transmembrane voltage (i.e. by feed-back        control) once the measured ionic current (I_(i)) has reached a        value (I_(p)) corresponding to the electrical conductance of a        pore in the TMDC thin layer having a prescribed diameter        (d_(p)).    -   (h) optionally replacing the electrically conducting liquid in        the cis chamber with another electrically conducting liquid        (e.g. suitable for nanopore biosensing).

Apart from nanopore sensors, other applications for a method accordingto the invention can be envisioned such as water desalination devicessuch as described in Yuan & Gaoquan, 2014, Nanoporous graphenematerials, Materials Today, 17 (2) 77-8 and Cohen-Tanugi Det al., 2012,2012, Nano Letters, 12, p. 3602-3608.

The invention having been described, the following examples arepresented by way of illustration, and not limitation.

EXAMPLES Example 1 Preparation of MoS₂ Nanopores Using ECR

The present example illustrates a method of the invention applied to thefabrication of individual nanopores on single-layer MoS₂, with theelectric field generated by Ag/AgCl electrodes located in twoelectrolytes compartments and positioned away from the membrane. Asdescribed below, the ECR process starts for a certain critical voltageapplied to the membrane at a defect/vacancy present in the MoS₂membrane.

Importantly, in the course of the ECR process, it is possible to controlthe successive removal of single or few MoS₂ units from the monolayerMoS₂ membranes. In this way, atom-by-atom nanopore engineering isachieved.

A procedure for fabricating MoS₂ nanopores using ECR is schematicallyillustrated in FIG. 2A, where two chambers (cis and trans) are filledwith aqueous buffer (1M KCl, pH 7.4) and biased by a pair of Ag/AgC1electrodes which are separated by a single-layer MoS₂ membrane. Presenceof an active site such as single-atom vacancy facilitates the removal ofindividual atoms and MoS₂ unit cells from MoS₂ lattice by ECR atvoltages higher than the oxidation potential of MoS₂ in aqueous media.This process is facilitated by the electric field focusing by the poreitself. To form freestanding membranes, CVD-grown monolayer MoS₂transferred from a sapphire substrate is suspended over focused ion beam(FIB) defined openings that ranged from 80 nm to 300 nm in diameter andwere centered in a 20 nm thick SiN_(x) membrane (FIG. 2B). A typicaloptical image of the transferred triangular flake of CVD-grown monolayerMoS₂ on the supporting silicon nitride membrane is shown in FIG. 2C. Thefreestanding MoS₂ membrane above the FIB defined opening can be furtheridentified under TEM with low magnification (5 k×) as shown in FIG. 2D.MoS₂ flake is further characterized by Energy-dispersive X-rayspectroscopy (EDX) in TEM to reveal the chemical composition of thesurface where elements of Mo and S are abundant in triangular areas.When moving to a high magnification (1 M×) and focusing on thefreestanding portion of MoS₂ over the FIB opening, the atomic structureof MoS₂ can be clearly resolved as shown in FIG. 2E, and thediffractogram reflects the hexagonal symmetry of MoS₂, as shown in theinset of FIG. 2E.

An intact MoS₂ membrane is mounted into a custom made microfluidicflow-cell filled with an aqueous buffer and transmembrane potential isapplied using a pair of Ag/AgCl electrodes as shown in FIG. 2A. When avoltage is applied below the potential for electrochemical oxidation ofthe transition metal of the membrane, small leakage current is normallydetected, typically on the order from tens to hundreds of picoamperesdepending on the number of defects in the 2D membrane. As shown in FIG.3A, the leakage current displays a non-ohmic characteristic. To reachthe critical voltage value for achieving an ECR, the potential isgradually stepped, as shown in FIG. 3B. When the applied voltage isstepped up to 0.8 V (a critical voltage, indicated by the arrow), anincrease of baseline current immediately occurs. This time-pointindicates the nanopore creation which is associated to theelectrochemical dissolution of MoS₂ enhanced by the electrical potentialfocused on the active site as shown in the potential profile obtained bythe finite element analysis simulation (FIG. 4A). In other words, thepore growth continues at the initial, active site instead of anotherstart on another defect because the pore itself focuses the appliedelectric potential and facilitates continuation of ECR on the activesite where ECR process has already started.

In contrast to the avalanche-like dielectric breakdown process insilicon nitride, where a typically 10-minute waiting time for thefilling of charge traps (Briggs et al., 2015, nanotechnology, 084004,doi:10.1088/0957-4484/26/8/084004) under the application of criticalvoltage (>10 V) is needed before breakdown occurs, electrochemicaldissolution happens spontaneously at the critical voltage.

In addition, the observed rise of ionic current shows a quite slow rate(˜0.4 nA/s). Therefore, it is possible to control the nanopore size byusing an automatic feedback to cut off the voltage once the desiredcurrent/conductance threshold is reached. This feedback also helps toavoid multiple pore formation. Owing to the limited rates ofelectrochemical reaction, the MoS₂ nanopore sculpting process is quiteslow, occurring on time scales of dozens of seconds to several minutes.FIG. 3E gives an example of ionic current trace to reach the thresholdof 20 nA, for the critical voltage of 0.8 V.

Taking the advantage of existing theoretical insights to model theconductance-pore size relation (Kowalczyk et al., 2011, Nanotechnology,22, doi:Artn 315101 Doi 10.1088/0957-4484/22/31/315101), the conductanceof the nanopore (G) can be described by

$\begin{matrix}{G = {\sigma \left\lbrack {\frac{4L}{\pi \; d^{2}} + \frac{1}{d}} \right\rbrack}^{- 1}} & (1)\end{matrix}$

where σ, L and d are the ionic conductivity of solution, membranethickness and nanopore diameter, respectively. Using this relation incombination with feedback on ECR that immediately stops the voltage oncethe desired pore conductance—that corresponds to a certain pore size—isreached, it is possible to fabricate pores ranging in diameter from 1-20nm. FIG. 3E reveals current-voltage (I-V) characteristics of MoS₂nanopores fabricated by ECR with different estimated sizes ranging from1 nm to 20 nm. The symmetric and linear I-V curves also imply thewell-defined shape of the fabricated pores. Similarly, as shown in theinset of the FIG. 3E, I-V characteristics across the membrane have beeninvestigated in situ before and after ECR, confirming the poreformation.

To further verify the size of fabricated MoS₂ nanopores, TEM has beenused to image the newly formed nanopore. Exposure of 2D materials toelectron radiation can induce large area damage and also open pores, asreported for both graphene (Fischbein et al., 2008, Applied PhysicsLetters, 93) and MoS2 (Liu et al., 2013, Nat. Commun., 4). Therefore,first aligned the beam was aligned on the suspended portion of MoS₂outside of the FIB opening and then quickly scanned the sample whiletaking care to minimally irradiate suspended MoS₂ that houses ECRfabricated pore. FIG. 2C shows a TEM image of an ECR-fabricated MoS₂nanopore.

For the sake of comparison, a few graphene membranes (prepared asdescribed below) have also been tested by this method and highervoltages (2-3V) are required to fabricate pores as presented inSupporting Information, with the typical ionic current trace isdisplayed in FIG. 4B.

The described ECR-based pore formation method benefits from the uniquecrystal structure of transition metal dichalcogenide (MX2) where atomsare situated in tree planes and linked by metal-chalcogenide bonds whilein the case of graphene, carbon atoms are in the same plane and 3 bondsneed to be removed to release one carbon atom. In addition, to removecarbon atoms, graphene needs to be oxidized to a higher valence statewhich presumably requires a higher voltage.

The physics of the electrochemically fabricated nanopores is determinedby the focused electrical field and surface chemistries. The electricfield concentrates at surface irregularities or defects which can beconsidered as surface active sites, and focuses current flow at the siteof the pore, and thus locally enhances the electrochemical dissolution,as shown in FIG. 3C.

The surface dissolution chemistries can be understood as a surface boundoxidation scheme with hole capture and electron injection to produce theMoS2 oxidation state (Bonde et al., 2008, Faraday Discussions 140,219-231) as shown in

MoS₂+11H₂O=MoO₃+2SO₄ ²⁻+22H⁺+18e ⁻  (2)

where MoS₂ is oxidized into MoO₃ which is water-soluble. Without beingbound to any theory, this reaction is highly likely to happenconsidering the electrical potential (voltage bias) range applied to themembrane. Due to the current technical limitations of electron energyloss spectroscopy (EELS) analysis in the nanopore vicinity, it cannot beexcluded the possibility that MoS₂ is oxidized to other valence states.Once an active site is removed by the process described above,indicating the initial formation of the pore, FIG. 5A, the electricfield, as simulated in FIG. 4A, entirely focuses in this pore, whichthen prefers to enlarge the same pore since this is more energeticallyfavorable than creating a second pore at another location. By applyingat the beginning of the fabrication process a bias voltage higher thanthe critical voltage it might be possible to observe the formation ofmultiple pores. Given the stochastic nature of the pore creationprocess, with a configuration of voltage steps, multiple simultaneousnanoscale ECR events are highly unlikely. Furthermore, feedback controlon the applied voltage to obtain the desirable conductance ensures theformation of a single nanopore. Finally, the formation of singlenanopore is confirmed by TEM imaging.

The advantage of an ECR-based nanopore fabrication technique of theinvention, apart from the benefit of being a fast and cheap productionlies in the possibility of fine-tuning the diameter of nanopores withunprecedented, single-atom precision. The low nanopore enlarging speedis due to low voltages and the electrochemical dissolution nature of theprocess. FIG. 5D is a 25-second long, continuous pore conductance tracethat shows atomic precision during nanopore sculpting process. The tracestarts from the critical point indicated in FIG. 3B. Fitting to theconductance-nanopore size relation, it can be estimated a pore diametergrowth rate of about 1 angstrom per second. After 25 seconds a pore witha diameter 1.9* nm (area of 2,9 nm²) has been formed. The area of such apore is equivalent to almost exactly N=34 unit cells of MoS₂ where thearea of the unit cell u=0.0864 nm² (FIG. 5A). *The final pore size is2,2, nm when one accounts for the initial 0.3 nm.

Surprisingly, the growth curve is not linear but step-like, as shown inFIG. 5B. Necessarily, the effective size of the pore enlarges with thesame step-like characteristic. These step-like features are commonlyobserved when working with voltages around 1V. To gain insights intothese step-like features, the histogram of current values is plottedfrom this trace in FIG. 5C, where 21 individual peaks can be extractedfrom the histogram.

The sequence of the pore size enlargement steps may be normalized by theunit cell area u and a sequence of MoS₂ formula units and Mo and S atomscleaved (corresponding to 21 current steps) to form the pore may beinferred, as presented in the FIG. 5C and 5E. Several snapshots of thepore formation process taken at steps 1,7,14 and 21 are displayed in theFIG. 5D. The area of polygons corresponding to the cleaved atom groupsfollows the honeycomb structure of single-layer MoS₂, as presented in anaberration corrected TEM image FIG. 5D and in the schematics shown inFIG. 5A. The observed atomic steps here reveal the ultimate precision(single atoms) that can be reached in engineering nanostructures with aprocess of nanopore forming of the invention.

Membrane Set-Up

The MoS₂ membranes are prepared using the previously reported procedure(Liu et al., 2014, ACS Nano 8, 2504-2511) and as described below.Briefly, 20 nm thick supporting SiN_(x) support membranes aremanufactured using anisotropic KOH etching to obtain 10 μm×10 μm to 50μ×50 μm membranes, with size depending on the size of the backsideopening. Focused ion beam (FIB) is used to drill a 50-300 nm opening onthat membrane. CVD-grown MoS₂ flakes were transferred from sapphiresubstrates using MoS₂ transfer stage in a manner similar to the widelyused graphene transfer method and suspended on FIB opening (Dumcenco etal., 2015, ACSNano., 9, 4611). Membranes are first imaged in the TEMwith low magnification in order to check suspended MoS₂ flakes on FIBopening.

CVD MoS₂ Growth

Monolayer MoS₂ has been grown by chemical vapor deposition (CVD) onc-plane sapphire. After consecutive cleaning byacetone/isopropanol/DI-water the substrates were annealed for 1 h at1000 ° C. in air. After that, they were placed face-down above acrucible containing ˜5 mg MoO₃ (≥99.998% Alfa Aesar) and loaded into afurnace with a 32 mm outer diameter quartz tube. CVD growth wasperformed at atmospheric pressure using ultrahigh-purity argon as thecarrier gas. A second crucible containing 350 mg of sulfur (≥99.99%purity, Sigma Aldrich) was located upstream from the growth substrates.Further details of the procedure are described in Dumcenco et al., 2014,supra.

CVD MoS₂ Transfer from Sapphire to SiN_(x) Membrane

Monolayer MoS₂ grown on sapphire substrate (12 mm by 12 mm) is coated byA8 PMMA (495) and baked at 180° C. A diamond scriber was used to cut itinto 4 pieces. Each piece is immersed into 30% w KOH at 85-90° C. forthe detachment. Capillary force might be used in the interface betweenpolymer and sapphire to facilitate the detachment and reduce the etchingtime in the KOH. The detached polymer film was repeatedly in DI water.Lastly, the “fishing” method of graphene transfer can be used totransfer CVD MoS₂ to the target SiN_(x) membrane.

Nanopore Forming

For the nanopore fabrication experiments, after mounting in thepolymethylmethacrylate (PMMA) chamber (FIG. 1B, step (a)), the chipswere wetted with H₂O: ethanol (v:v, 1:1) for at least 20 min (FIG. 1B,step (b)). 1 M KCl solution buffered with 10 mM Tris-HCl and 1 mM EDTAat pH 8.0 was injected in the chamber (FIG. 1B, step (c)). A pair ofchlorinated Ag/AgCl electrodes was immersed in the chamber (FIG. 1B,step (d)) and employed to apply the transmembrane voltage and thecurrent between the two electrodes was measured by a FEMTO DLPCA-200amplifier (FEMTO® Messtechnik GmbH). A low voltage (100 mV) was appliedto check the current leakage of the membrane. If the leakage current wasbelow 1 nA, the voltage bias was set up in 100 mV steps (25 s for eachstep) FIG. 1B, step (e)). At a critical voltage (i.e. 800 mV), thecurrent started to immediately increase above the leakage level. A FPGAcard and custom-made Lab View software was used for applying the voltageand monitoring the conductance (FIG. 1B, step (f)). The critical voltagewas automatically shut-down by a feedback control implemented in LabViewprogram as soon as the desirable conductance was reached (FIG. 1B, step(g)). Nanopores were further imaged using a JEOL 2200FS high-resolutiontransmission electron microscope (HRTEM). STEM-EDX was performed on aChemiSTEM-equipped FEI Tecnai Osiris transmission electron microscope(TEM). Aberration corrected TEM micrographs were taken on FEI TitanThemis.

CVD Graphene Growth for Comparative Examples

Large-area graphene films are grown on copper foils. The growth takesplace under the flow of a methane/argon/hydrogen reaction gas mixture ata temperature of 1,000° C. At the end of the growth, the temperature israpidly decreased and the gas flow turned off. The copper foils are thencoated by PMMA and the copper etched away, resulting in a cm-scalegraphene film ready to be transferred on the chips with membranes forfabricating nanopores with a method of the invention.

Finite Element Analysis Model

To estimate the potential drop in a defect in a MoS₂ membrane a finiteelement analysis was performed using COMSOL Multiphysics 4.4b. A coupledset of the Poisson-Nernst-Planck equations was solved in a 3D geometrywith axial symmetry. In the modeled configuration cis and trans chamberswere connected by a 0.3 nm pore in a 0.7 nm thick membrane suspended ona 50 nm wide and 20 nm thick hole. A 0.3 nm diameter defect cancorrespond to the absence of a unit cell of MoS₂. In the model, theapplied potential was set to 800 mV and salt concentration was 1 M KCl.The minimal mesh size used was less than 0.2 Å.

Detailed Data Analysis of Ionic Current Steps Presented in FIGS. 5A-E

All analysis were implemented in Matlab R2014b. The raw signal wasdown-sampled to 5 kHz and then filtered using the edge-preservingChung-Kennedy (CK) filter (Chung et al., 1991, J. Neurosci. Methods, 40,71-86). The pore formation in 21 steps, presented on FIGS. 5A-E can beas follows: the growth of the nanopore is due to sequential cleaving ofunit cells from MoS₂ lattice. The final pore area is 2.9 nm², whichcorresponds to 34 unit cells. Increments in the effective pore size AAare normalized by unit cell size u=0.0864 nm². The obtained number AN=AA/u was rounded to the nearest integer, integer +⅓ or integer + 2/3 toget ΔX, the number of MoS₂ unit cells cleaved during the pore formationprocess. It is assumed that ⅓ corresponds to a S₂ group and ⅔ to a Moatom, corresponding to the partial cleaving of a unit cell. It should benoted that the two S atoms in MoS₂ are stacked vertically and theircombined surface area is smaller than that for Mo (which has about 50%larger radius).

The sequence of cleaving MoS₂ unit cells and Mo and S atoms in 21 stepsto form the pore is given in the table from FIG. 5E. In order to depictthe sequence of the pore formation, the numbering of the lines in thistable and polygons based on HRTEM image starts from 1 to 21. Lifetime ofthe steps in the sequence is given in the same table. Initiallyirregular pore gradually becomes more symmetrical. The atom groups havebeen selected in the manner to minimize the number of dangling bonds atthe edge of the pore. The pore formation sequence is not unique,however, the dangling bond constraint significantly reduces the numberof pore formation scenarios and induces more symmetrical pore shape.

Example 2 DNA Sensing with Nanopores Fabricated by a Method of theInvention

To test the performance of ECR-fabricated pores, DNA translocationexperiments were carried and detected the translocation events by thecurrent drops below the baseline current. ECR fabricated MoS₂ nanoporesconsistently produces low-1/f noise on the current baseline in the rangeof 50-100 pA. The major contribution to the 1/f noise in 2D membranenanopores can be attributed to mechanical fluctuations of the thinmembranes. Higher frequency fluctuations are produced by the methoditself. Fluctuation noise can be significantly reduced by using asmaller supporting opening, or operating at low temperatures. To showthe ability of ECR fabricated nanopore for DNA detection, 2.7 kbp pNEBplasmid DNA (New England Biolabs) is translocated through a relativelylarge MoS₂ nanopore (25 nm) to eliminate the pore-DNA interaction andmultiple conformation issues. FIG. 6A displays only one-level eventsindicating an extended (unfolded) DNA conformation, with SNR>10. Scatterplots are used to describe the statistics of DNA translocation as shownin FIG. 6B. The signal amplitude also increases linearly with theapplied voltage, which is 0.5 nA for 450 mV and 0.38 nA for 300 mV asshown in the histogram FIG. 6B. Dwell times are also comparable with DNAtranslocation through a TEM-drilled MoS₂ nanopore of a similar diameter,for the same DNA and under same bias conditions. In addition, k-DNA (48k bp) is also translocated through an ECR-fabricated nanopore. Asexpected, folding scenario can be observed, manifested by quantizationof current levels guided by dash lines. The upper dash line is the onelevel conductance, indicating a linear translocation. The bottom dashline is the two level conductance, indicating a folded translocation(FIG. 7).

Nanopore Testing for DNA Sensing

Current-voltage (I-V) characteristic and DNA translocation were recordedon an Axopatch 200B patch clamp amplifier (Molecular Devices, Inc.Sunnyvale, Calif.). DNA samples (pNEB193, plasmid 2.7 k bp, New England;k-DNA, 48 k bp, New England) were diluted by mixing 10 μL of λ-DNA orpNEB stock solution with 490 μL 1 M KCl buffer. NI PXI-4461 card wasused for data digitalization and custom-made LabView software for dataacquisition using Axopatch 200B. The sampling rate is 100 kHz and abuilt-in low-pass filter at 10 kHz is used. Data analysis enabling eventdetection is performed offline using a custom open source Matlab code,named OpenNanopore (Raillon et al, Nanoscale, 2012, 4, 4916-4924)(lben.epfl.ch/page-79460-en.html).

Altogether, those data support that a method of the invention leads toreproducible and fully characterized nanopores by I-V characteristicsand size as confirmed by TEM. Further, the intrinsic electrochemicalreaction kinetics permits an advantageously high precision for nanoporefabrication at the atom level which can be monitored by the observedstep-like features in the ionic current traces. Finally, the nanoporesobtained through a method of the invention have demonstrated to allowDNA translocation and their sensing as fully functional nanopores.

Example 3 Preparation of WSe₂ Nanopores Using ECR

The present example illustrates a method of the invention applied to thefabrication of individual nanopores on single-layer of tungstendiselenide (WSe₂), with the electric field generated by Ag/AgClelectrodes located in two electrolytes compartments and positioned awayfrom the membrane. Exfoliated single layer of WSe₂ has been transferredto silicon nitride membrane and positioned over the small opening on thesame device layout as MoS₂ and as described in Example 1 (FIG. 9A)).This sample is mounted to the flow cell filled with 1M KCl (pH 11).Trans-membrane voltage is stepped gradually to reach critical voltage. Ananopore is formed at 1V and IV current voltage is taken immediatelyafter pore formation and current voltage characteristics of nanoporeformed with ECR in WSe₂ are similar to those of current voltage obtainedwith MoS₂ (FIG. 9B)) Representative ionic current trace measured forWSe₂ membrane. The corresponding ionic current trace measured for avoltage set to the critical voltage of 1V is represented under FIG. 9C)and the current keeps increasing until desired pore size is reached.

Constant Current Nanopore Formation Apparatus for Controlling Current

In some embodiments, current may be held at a constant level in forminga nanopore of a given diameter, which may result in better control ofthe final diameter of the nanopore because the initial opening of anaperture significantly decreases the resistance in a circuit.

The apparatus for forming a nanopore before an aperture is defined isshown in FIGS. 10A and 10B. FIG. 10A shows a top view of apparatus 400according to embodiments. FIG. 10B shows a side view of apparatus 400.These figures are not drawn to scale. A layer of transition metaldichalcogenide crystals 402 contacts insulating material 404. Theinsulating material may define an orifice 406. Orifice 406 may bepatterned by e-beam and the reactive ion etching. Orifice 406 may have adiameter in a range from about 15 nm to about 80 nm. Orifice 406 mayhave sidewalls that are straight or tapered depending on the patterningtechnique.

On one side of layer of transition metal dichalcogenide crystals 402 maybe a first liquid 408. First liquid 408 may be in a sealed compartment,such that no liquid can flow in or out of the sealed compartment. On theother side of layer of transition metal dichalcogenide crystals 402 maybe a second liquid 410. Second electrically conductive liquid 410 mayalso be in a sealed compartment. First liquid 408 and second liquid 410may be isolated from each other such that they are not in fluidcommunication, at least before the nanopore is formed. One electrode 412may be in first liquid 408, and another electrode 414 may be in secondliquid 410.

A power supply 420, also called a voltage source, can provide a voltageacross electrodes 412 and 414. A circuit can be formed among wiresconnecting electrodes 412 and 414 and the power supply. The circuit mayinclude other components, e.g., for a current measuring device formeasuring current in the circuit, as will be known to one skilled in theart. A control circuit (not shown) may exist in power supply 420 or as aseparate component for varying the voltage applied to the electrodes.For example, the control circuit can vary the voltage so that a currentin the circuit is constant.

As a result of the voltage applied to electrodes 412 and 414, anaperture may be created in the layer of transition metal dichalcogenidecrystals 402, and a current can flow between electrodes 412 and 414through the first and second liquids. Each electrode may have alongitudinal axis. The longitudinal axis may be aligned with the lengthof the electrode and in the same direction as the length of theelectrode. In some embodiments, the longitudinal axis may be a lineabout which the electrode is symmetric. For example, the longitudinalaxis may be a line that bisects the electrode in the same direction asthe length of the electrode. The longitudinal axis or a line coincidentwith the longitudinal axis may extend through orifice 406. At least oneof the longitudinal axes of electrodes 412 and 414 may point towardorifice 406. A line having the shortest distance between electrode 412and electrode 414 may pass through orifice 406 and/or an aperture thatis formed later.

The current may flow from electrode 412 through first liquid 408,through layer of transition metal dichalcogenide crystals 402, throughsecond liquid 410, and to electrode 414, or flow in an oppositedirection. One electrode may be a ground electrode. Electrodes 412 and414 may each be a few microns away from the layer of transition metaldichalcogenide crystals. As mentioned above, both electrodes 412 and 414are in electrical communication with power supply 420, which may beconfigured to deliver at least one of a constant voltage or a constantcurrent. The electrical circuit may include a control system formaintaining current at a constant level. The control system may includea processor.

Apparatus 400 may be one of a plurality of apparatuses for usingnanopores to detect or analyze biological molecules. The plurality ofapparatuses may include thousands to millions of apparatuses configuredfor multiplex analysis. The apparatuses may exist as an array ofnanopores. A single ground electrode may exist, with each nanoporehaving a separate non-ground electrode.

FIGS. 10C and 10D show the apparatus after an aperture has been defined.FIG. 10C shows a top view of apparatus 400, and FIG. 10D shows a sideview of apparatus 400. An aperture 416 has formed in layer of transitionmetal dichalcogenide crystals 402. This aperture may be formed after acritical voltage is reached.

The formation of the aperture affects the current-voltagecharacteristics of the apparatus and may present certain advantages tomaintaining a constant current. Voltage is related to current by Ohm'sLaw:

V=IR

where Vis voltage, I is current, and R is resistance. Before an apertureis formed, the resistance of the circuit may be high. While liquids 408and 410 may be conductive, layer of transition metal dichalcogenidecrystals 402 may be a semiconductor. Once an aperture is formed, even ifthe diameter of the aperture is still small, an electrical path may passthrough the electrically conductive liquid in the aperture, and theresistance of the circuit may decrease.

In scenarios where the voltage is increased gradually or stepwise, asshown in FIG. 3B, the current may be zero or near zero until the voltagereaches a critical voltage. At that critical voltage, an aperture forms.The resistance decreases with the opening of the aperture. If thevoltage remains constant after the aperture forms, then the currentincreases and the aperture may continue to widen. The current mayincrease quickly, and the aperture may widen quickly. With such rapidincreases in current, turning off the voltage at the exact moment thetarget diameter of the nanopore is reached may be difficult. As aresult, the diameter of the nanopore may overshoot the target diameter.

In contrast, scenarios involving a current at a constant levelthroughout the formation of the aperture may result in improved controlof the target diameter. Even before an aperture is formed, the voltagecan be periodically adjusted to maintain current at a constant level(e.g., within a rated tolerance provided by the power supply). With noaperture, a large voltage may be generated. After the aperture isinitially formed and the resistance decreases, the voltage may alsodecrease proportionally in order to maintain the current at a constantlevel. The current may be set at a level corresponding to theconductance of the electrically conductive liquids and a predetermined,target diameter of the nanopore. Once the diameter of the aperturematches the target diameter of the nanopore, the resistance and thevoltage may remain constant, and the aperture may no longer widen. As aresult, the voltage may not need to be decreased immediately when thetarget diameter is reached, and therefore, improved control in reachingthe target diameter may be achieved. Indeed, both before and after theaperture is formed, the system may act to maintain the constant currentby adjusting voltage. Because the control scheme remains largely thesame, applying a constant current may also permit process simplicity andefficiency. Applying a constant current may involve different equipmentthan equipment used for applying a voltage with a variable current.

An aperture formed may be configured to have a subunit of a polymermolecule disposed within the aperture. A subunit of a polymer moleculedisposed in the aperture may change a current between the firstelectrode and the second electrode. The change in current may bedetectable, indicating the presence of the subunit of the polymermolecule.

Method

As shown in FIG. 11, embodiments may include a method 1100 of forming ananopore in a layer of transition metal dichalcogenide crystals.

At block 1102, a variable voltage may be applied across a firstelectrode and a second electrode such that an aperture is created in thelayer of transition metal dichalcogenide crystals.

The layer of transition metal dichalcogenide crystals may be between afirst electrode and a second electrode, with the first electrode on oneside of the layer and the second electrode on another side of the layer.For example, the first electrode may be on an opposite side of the layeras the second electrode.

A first liquid may be on one side of the layer of transition metaldichalcogenide crystals, and a second liquid may be on another side ofthe layer of transition metal dichalcogenide crystals. The first liquidmay include aqueous, organic, or ionic liquid and may be electricallyconductive. The first liquid may include ions formed from a salt. Thesalt may be an inorganic salt and may include one selected from thegroup consisting of KCl, LiCl, NaCl, and MgCl₂. In addition, the firstelectrically conductive liquid may include a room temperature ionicliquid (RTIL). The second liquid may be any of the liquids listed forthe first liquid. In some embodiments, the second liquid may have a sameor different composition from the first liquid.

The transition metal dichalcogenide crystals may include a compoundhaving a chemical formula MX₂, where M is a transition metal atom, and Xis selected from the group consisting of sulfur, selenium, andtellurium. Transition metal atoms may include Ta, Nb, Mo, W, Ti, and Re.The transition metal dichalcogenide crystals may include a compoundselected from the group consisting of MoS₂, SnSe₂, WS₂, TeS₂, MoSe₂,WSe₂, TeSe₂, NbS₂, NbSe₂, TiS₂, TiSe₂, ReS₂, and ReSe₂.

The layer may be one monolayer thick, two monolayers thick, or threemonolayers or more thick. The thickness of the layer may range fromabout 0.3 nm to about 5 nm. A monolayer may include the transition metalatom sandwiched by two planes of chalcogenide crystals. For example, asshown in FIG. 5A, a molybdenum atom may be between two planes of sulfuratoms. Based on this structure, the layer may be three atoms thick. Thecrystalline structure of the layer may allow for predictable orquantifiable effects at the edges of the layer, where a nanopore islater formed.

The layer of transition metal dichalcogenide crystals may be contactingan insulating material. The insulating material may include siliconnitride, glass, Al₂O₃, HfO₂, quartz, or TiO₂. The insulating materialmay define an orifice.

The aperture may form within the orifice defined by the insulatingmaterial. In other words, a line going through the center of the orificeand orthogonal to the insulating material may extend through theaperture.

At block 1104, method 1100 may include varying the variable voltage asthe aperture widens such that a current having the constant averagecurrent level exists between the first electrode to the secondelectrode. The current may be an alternating current or a directcurrent. Direct current may be considered to have zero amplitude andzero frequency. The current may have a constant average current levelcorresponding to a specified diameter of the aperture to be formed. Theresistance of the circuit may drop as the aperture forms. The currentmay be maintained at a constant average current level by adjusting thevariable voltage. The formation of the aperture may be detected bymeasuring a drop in the variable voltage. As examples, the averagecurrent level for an alternating current may be defined as the averagecurrent for one cycle or as a root mean square average. With alternatingcurrent, a constant average current level can result from a constantamplitude for the current. The current applied may be in the fA, pA, nA,μA, mA, or A range.

At block 1106, method 1100 may include maintaining the current at theconstant average current level such that the aperture widens to thespecified diameter. After the aperture forms, the current may existthrough the aperture instead of through the layer of transition metaldichalcogenide crystals. In some embodiments, the aperture may continueto widen past the specified diameter even if average current level isnot increased, but the aperture may have the specified diameter at somegiven time. In some embodiments, chemical entities may be added to theliquids on either one or both sides to react with the molecular entitiesconstituting the edge of the layer of transition metal dichalcogenidecrystals defining the widening aperture.

Method 1100 may further include decreasing the variable voltage tomaintain the current at the constant average current level after theaperture having the specified diameter is formed. Analysis of thevoltage, resistance, or the duration of current applied may helpindicate when the specified diameter is reached. After the specifieddiameter is reached, the current may no longer be maintained at aconstant average current level. The current may be decreased bydecreasing the voltage. Decreasing the voltage may include decreasingthe voltage to 0 V. Decreasing the voltage may include turning off thevoltage as rapidly as possible. Decreasing the voltage may includedecreasing the voltage to a level suitable for detection or analysis ofa polymer molecule (e.g., when the polymer molecule is in linear form)in the nanopore. In some embodiments, method 1100 may include maintainvoltage at a constant level after the aperture having the specifieddiameter is formed.

At block 1108, method 1100 may further include providing a polymermolecule in the nanopore. The polymer molecule may be introduced intothe first or second liquid. Method 1100 may also include applying avoltage across the two electrodes and/or may include providing a currentbetween the two electrodes.

At block 1110, method 1100 may further include detecting a subunit of apolymer molecule in the nanopore. The charged electrodes may driveanions in the solution to the positive electrode and cations in thesolution to the negative electrode, creating an ionic current. If asubunit of a polymer molecule enters the nanopore, it may disrupt theionic flow. The subunits of the polymer molecule may be detected bymeasuring a change in the ionic current. Polymer molecules may includeDNA, single-stranded DNA, RNA, proteins, and non-native polymers.Non-native polymers may include a molecule such as DNA with anartificial tag inserted in the molecule to make the molecule easier tobe detected or analyzed by the nanopore. As examples, the subunit of thepolymer molecule may include a base or a plurality of bases for nucleicacids or amino acid(s) for proteins. Different subunits may havedifferent effects (e.g., amplitude and dwell time) on the current, andas a result, different subunits may be identified.

The liquids, layer of transition metal dichalcogenide crystals, voltage,current, aperture, nanopore, and electrodes may be any described herein.

Applied Voltage with Variable Current Nanopore Formation

Method

As shown in FIG. 12, embodiments may include a method 1200 of forming ananopore in a layer of transition metal dichalcogenide crystals.

At block 1202, a voltage at a first voltage level may be applied acrossa first electrode and a second electrode and across the layer oftransition metal dichalcogenide crystals such that an aperture iscreated in the layer of transition metal dichalcogenide crystals.Previous to the first voltage level being achieved, the voltage may beapplied in a stepwise or increasing manner until the voltage reaches thefirst voltage level corresponding to the formation of the aperture. Thefirst voltage level may be higher than the oxidation potential of thetransition metal of the transition metal dichalcogenide crystals. Thevoltage may be at a level in a range from about 800 mV to 1,000 mV. Thevoltage may include a direct current voltage or an alternating currentvoltage.

At block 1204, the voltage may be maintained at the first voltage levelfor a specified amount of time. The specified amount of time maycorrespond to the aperture widening to have at least a specifieddiameter. As examples, the specified amount of time may be determined bymeasuring the time to achieve a given average current, or the specifiedamount of time may be based on average times for apertures to be createdwith the specified diameter in previous samples. When previous samplesare used, the amount of time can be determined from a statisticaldistribution of the times (e.g., as determined by measuring current) tocreate the nanopores with the specified diameter. The amount of time touse can be based on various statistical parameters of the statisticaldistribution, e.g., the time can be taken as the average, mode, median,specific percentiles (e.g., 90% of nanopores have at least the specifieddiameter at the given amount of time), or standard deviations.

At block 1206, the voltage may be reduced to a second voltage level toinhibit the widening of the aperture. Method 1200 may include measuringa current through the first liquid and the second liquid. The currentmay be reduced after the current reaches an average current levelcorresponding to the specified diameter of the aperture. Reducing thevoltage may include activating a switch through a feedback controlcircuit when the current level is reached. The aperture may continue towiden past the specified diameter even after the voltage is reduced.However, the aperture may eventually stop widening and reach a maximumdiameter. The second voltage level may be non-zero.

At block 1208, method 1200 may include providing a polymer molecule inthe nanopore. The polymer molecule may be provided similar to othermethods described herein.

At block, 1210, a subunit of the polymer molecule in the nanopore may bedetected, similar to other detection methods described herein.

The liquids, layer of transition metal dichalcogenide crystals, voltage,current, aperture, nanopore, and electrodes may be any described herein.

What is claimed is:
 1. A method of forming a nanopore in a layer oftransition metal dichalcogenide crystals residing between a firstelectrode on a first side of the layer of transition metaldichalcogenide crystals and a second electrode on a second side of thelayer of transition metal dichalcogenide crystals, wherein a firstliquid is disposed on the first side and a second liquid is disposed onthe second side, the method comprising: applying a voltage at a firstvoltage level across the first electrode and the second electrode andacross the layer of transition metal dichalcogenide crystals such thatan aperture is created in the layer of transition metal dichalcogenidecrystals; and maintaining the voltage at the first voltage level for aspecified amount of time, the specified amount of time corresponding tothe aperture widening to have at least a specified diameter; after thespecified amount of time, reducing the applied voltage to a secondvoltage level to inhibit the widening of the aperture.
 2. The method ofclaim 1, wherein: the transition metal of the transition metaldichalcogenide crystals has an oxidation potential, and the firstvoltage level is higher than the oxidation potential.
 3. The method ofclaim 1, further comprising measuring a current between the firstelectrode and the second electrode, and wherein reducing the appliedvoltage comprises reducing the applied voltage after the current reachesa current level corresponding to the specified diameter of the aperture.4. The method of claim 3, wherein reducing the voltage comprisesactivating a switch through a feedback control circuit when the currentlevel is reached.
 5. The method of claim 4, wherein the activation ofthe switch turns off the applied voltage.
 6. The method of claim 1,wherein the first voltage level is in a range from 800 mV to 1,000 mV.7. The method of claim 1, wherein applying the voltage comprisesapplying a direct current voltage.
 8. The method of claim 1, wherein thetransition metal dichalcogenide crystals comprise a compound having achemical formula MX₂, wherein M is a transition metal atom, and whereinX is selected from a group consisting of sulfur, selenium, andtellurium.
 9. The method of claim 1, wherein the transition metaldichalcogenide crystals comprises a compound selected from the groupconsisting of MoS₂, SnSe₂, WS₂, TeS₂, MoSe₂, WSe₂, TeSe₂ NbS₂, NbSe₂,TiS₂, TiSe₂, ReS₂, and ReSe₂.
 10. The method of claim 1, wherein thelayer of transition metal dichalcogenide crystals is disposed on aninsulating material.
 11. The method of claim 1, wherein: the firstelectrode is disposed in the first liquid, and the second electrode isdisposed in the second liquid.
 12. The method of claim 1, wherein thelayer of transition metal dichalcogenide crystals has a thickness in arange from 0.3 nm to 5 nm.
 13. The method of claim 1, wherein the layerof transition metal dichalcogenide crystals is one monolayer thick. 14.The method of claim 1, wherein the layer of transition metaldichalcogenide crystals is two monolayers thick.
 15. The method of claim1, wherein the specified diameter is in a range from 1 nm to 5 nm. 16.The method of claim 1, further comprising detecting a subunit of apolymer molecule in the nanopore based on electrical signals detected byat least one of the first electrode and the second electrode.
 17. Themethod of claim 1, wherein the specified amount of time is based onaverage times for apertures to be created with the specified diameter inprevious samples.